1. Field of the Invention
The present invention relates to molecular sensors and methods related thereto.
2. Background
The development of various methods for nucleic acid detection and the detection of nucleic acid amplification products has led to advances in the detection, identification, and quantification of nucleic acid sequences in recent years. Nucleic acid detection is potentially useful for both qualitative analyses, such as the detection of the presence of defined nucleic acid sequences, and quantitative analyses, such as the quantification of defined nucleic acid sequences. For example, nucleic acid detection may be used to detect and identify pathogens; environmental monitoring; detect genetic and epigenetic alterations that are linked to defined phenotypes; diagnose genetic diseases or the genetic susceptibility to a particular disease; assess gene expression during development, disease, and/or in response to defined stimuli, including drugs; as well as generally foster advancements in the art by providing research scientists with additional means to study the molecular and biochemical mechanisms that underpin cellular activity. See U.S. Pat. No. 7,226,738.
In 1953, Watson and Crick suggested the concept of double stranded DNA. They had some significant discoveries. (1) deoxyribonucleic acid (DNA) molecules were composed of two anti-parallel poly-nucleic acid chains. (2) There were rules for paring the four bases—Chargaff et al. analyzed the base compositions of DNA molecules by chromatograph from many organisms, and found that the numbers of A and T were equal, while the numbers of C and G were also equal. So they suggest there exist four possible base pairs: A-T, T-A, G-C and C-G. (3) The connection of the two chains were through hydrogen bounds—the surface of the base pairs goes through and was roughly perpendicular to the axis. Two and three hydrogen bounds can form between the A-T and G-C pairs, respectively. Meanwhile, hydrophobic force also contributes to stabilize the DNA double helixes. (4) Because all the base pairs follow these rules, every chain can have random sequences. However, once the sequence of one of the chains is determined, the other one must have the corresponding nucleotide sequences. See U.S. Pat. No. 7,101,671.
As the DNA double helix is maintained by hydrogen bonds and hydrophobic force, factors, such as heat, pH, organic solvent, etc., which can destroy hydrogen and hydrophobic bonds, thus denaturing DNA double helixes to random single chain threads. The annealing between denatured DNA single chains through pairing is called hybridization. Hybridization can occur between homologous DNA molecules as well as homologous DNA and ribonucleic acid (RNA) molecules. During hybridization, the two complementary single-stranded DNA chains form double-stranded hybrids through non-covalent bounds. When the sequence of one of the chains is known, the existence of its complementary chain in an unknown DNA sample can be detected through hybridization. See U.S. Pat. No. 7,101,671.
Nucleic acid detection technology generally permits the detection of defined nucleic acid sequences through probe hybridization, that is, the base-pairing of one nucleic acid strand with a second strand of a complementary, or nearly complementary, nucleic acid sequence to form a stable, double-stranded hybrid. Such hybrids may be formed of a RNA segment and a DNA segment, two RNA segments, or two DNA segments, provided that the two segments have complementary or nearly complementary nucleotide sequences. (As is well known, a molecule of DNA or RNA possesses directionality, which is conferred through the 5′ 3′ linkage of the sugar-phosphate backbone of the molecule. Two DNA or RNA molecules maybe linked together through the formation of a phosphodiester bond between the terminal 5′ phosphate group of one molecule and the terminal 3′ hydroxyl group of the second molecule. See U.S. Pat. No. 6,933,121.
A known method for gene analysis to analyze DNA sequences in non-homogenous system is through DNA hybridization. Under sufficiently stringent conditions, nucleic acid hybridization may be highly specific, requiring exact complementarily between the hybridized strands. Typically, nucleic acid hybrids comprise a hybridized region of about eight or more base pairs to ensure the binding stability of the base-paired nucleic acid strands. Hybridization technology permits the use of one nucleic acid segment, which is appropriately modified to enable detection, to “probe” for and detect a second, complementary nucleic acid segment with both sensitivity and specificity. In the basic nucleic acid hybridization assay, a single-stranded target nucleic acid (either DNA or RNA) is hybridized, directly or indirectly, to a labeled nucleic acid probe, and the duplexes containing the label are quantified. Both radioactive and non-radioactive labels have been used. See U.S. Pat. No. 7,226,738.
Several methods have been advanced as suitable means for detecting the presence of low levels of a target nucleic acid in a test sample. One category of such methods is generally referred to as target amplification, which generates multiple copies of the target sequence, and these copies are then subject to further analysis, such as by gel electrophoresis, for example. Other methods generate multiple products from a hybridized probe, or probes, by, for example, cleaving the hybridized probe to form multiple products or ligating adjacent probes to form a unique, hybridization-dependent product. Still other methods amplify signals generated by the hybridization event, such as a method based upon the hybridization of branched DNA probes that have a target sequence binding domain and a labeled reporting sequence binding domain. See U.S. Pat. No. 7,226,738.
Techniques have been developed recently to meet the demands for rapid and accurate detection of pathogens, such as bacteria, viruses, parasites, and fungi, for example, as well as the detection of normal and abnormal genes. While all of these techniques offer powerful tools for the detection and identification of minute amounts of a target nucleic acid in a sample, they all suffer from various problems. For example, recognized disadvantage associated with current nucleic acid probe technologies is the lack of sensitivity of such assays when the target sequence is present in low copy number or dilute concentration in a test sample. In many cases, the presence of only a minute quantity of a target nucleic acid must be accurately detected from among myriad other nucleic acids that may be present in the sample. The sensitivity of a detection assay depends upon several factors: the ability of a probe to bind to a target molecule; the magnitude of the signal that is generated by each hybridized probe; and the time period available for detection. Another recognized disadvantage associated with current nucleic acid probe technologies is the reliance on fluorescence or radioactivity for nucleic acid detection.
Specifically, gene-probe assays may use a label that is either toxic or requires substantial expertise and labor to use. Radiolabeling is one of the most commonly used techniques because of the high sensitivity of radiolabels. But the use of radiolabeled probes is expensive and requires complex, time consuming, sample preparation and analysis and special disposal. Alternatives to radioactivity for labeling probes include chemiluminescence, fluorimetric and colorimetric labels, but each alternative has distinct disadvantages. Colorimetry is relatively insensitive and has limited utility where minute amounts of sample can be obtained. Samples must also be optically transparent. Fluorimetry requires relatively sophisticated equipment and procedures not readily adapted to routine use. Chemiluminescence, although versatile and sensitive when used for Southern blots, northern blots, colony/plaques lift, DNA foot-printing and nucleic acid sequencing, is expensive, and is not well-adapted for routine analysis in the clinical laboratory.
Other disadvantages of conventional sensors, such as gel micro-arrays, include difficulties with optical read-out, which include difficulties with aligning fluorescence spots with pads, and distinguishing a large a background signal from a signal of interest. Moreover, fluorescent tagging requires additional sensitive steps (after isolating target molecules from a sample, it must be tagged with a fluorescent molecule. In addition the instruments are bulky and expensive.
Accordingly, there is a need for rapid, sensitive, and standardized nucleic acid detection apparatuses and methods that can detect low levels of a target nucleic acid in a test sample that do not rely on fluorescence or radioactivity. These needs, as well as others, are met by the inventions of this application.